vaccine manufacture. R. Levis We have shown that the first nonstructural protein in the DEN2 genome, NS1, can trans complement lethal mutations of this protein in the full-length "infectious" cDNA clone. We have continued studies on the role of NS1 in virus replication and spread. One mutation in NS1 which removes 25 amino acids at the carboxy-terminus of the NS1 open reading frame was lethal for virus replication in control cells and was not complemented in trans in NS1 expressing cells. This mutation defines a region that is cis-essential for virus replication. We are currently constructing subclones of this region to determine what sequences are required for the cis-activity of this region. We hypothesize that this region may affect proper processing of downstream viral proteins. In vitro and in vivo processing assays are currently being developed to study this observation. We have also continued studies on the enhanced growth of wildtype dengue 2 virus in LLC-MK2 cells expressing dengue 2 NS1. Initial studies on NS1 expressing cells infected with dengue 2 showed that the NS1 expressing cells were resistant to virus induced apoptosis. Further studies on virus induced apoptosis in control and NS1 expressing cells show that NS1 expressing cells are resistant to apoptosis induced by C2-ceramide. We have initiated further biochemical studies to understand the interaction between NS1 and the cellular proteins which regulate apoptosis. One aspect of these studies was to measure the interferon response of control and NS1 expressing cells after infection with dengue virus. Initial studies show that only the non-NS1 expressing, control cells produce interferon after infection with dengue 2 or after treatment with polyI:C. We are currently characterizing the interferon response in these and NS1 expressing cells. We have initiated a new study on the role of the dengue virus NS4B protein in virus replication. We have established a system similar to the one previously described for NS1 expression in which we have created an LLC-MK2 cell line expressing dengue 2 NS4B. However, lethal mutations in NS4B cannot be complemented in trans using this cell line. This suggests that NS4B must function in cis in the viral genome, and/or must form complexes with other dengue nonstructural proteins which cannot be made by NS4B provided in trans. We are currently looking at potential interactions of NS4B with other viral proteins by coimmunoprecipitation and by coexpression studies.